Neublastin is a secreted protein which promotes the survival of neurons of the peripheral and central nervous system such as dopaminergic neurons (Baudet et al., 2000, Development, 127:4335; Roseblad et al., 2000, Mol. Cell Neurosci., 15(2):199; GenBank AF120274). The gene encoding neublastin has been cloned and sequenced (Roseblad et al., 2000, Mol. Cell Neurosci., 15(2):199; Baloh et al., Neuron, 21:1291).
Neublastin is a member of the glial cell line-derived neurotrophic factor (GDNF) ligand family. At the cellular level, GDNF members activate the receptor tyrosine kinase, RET. RET associates with a co-receptor, GDNF family receptor α (GFR α), a glycosylphosphatidyl inositol (GPI) linked membrane protein that provides ligand specificity for RET. Four GFRα's are known (GFRα1-4). Neublastin binds to GFRα3, (Baudet et al. 2000, Development, 127:4335; Baloh et al., 1998, Neuron, 21:1291) which is expressed predominantly in nociceptive sensory neurons (Orozco et al., 2001, Eur. J. Neurosci., 13(11):2177). These neurons detect pain and injury. Thus, neublastin has clinical application in the general treatment of neuropathy and more specifically in the treatment of pain.
Neublastin and the other GDNF family members are distant members of the transforming growth factor β (TGF β) superfamily and thus, are characterized by the presence of seven conserved cysteine residues with similar spacing which form the structure of a cysteine knot (Saarma, 1999, Microsc. Res. Tech., 45:292). The cysteine knot is comprised of a loop formed by two disulfide bridges through which a third disulfide bond passes (Rattenholl et al 2000, J. Mol. Biol., 305:523).
TGF β family members are synthesized as pre pro proteins that eventually are secreted as a mature homodimer after cleavage of the signal peptide and pro-domain (see e.g. Rattenholl, et al., 2000, J. Mol. Biol., 305:523; Fairlie et al., 2001, J. Biol. Chem., 276(20):16911). The signal peptide mediates secretion. The pro-domain mediates proper secretion for TGF β family members (Rattenholl et al., 2000, J. Mol. Biol., 305:523; Rattenholl et al., 2001, Eur. J. Biochem., 268:3296). Although macrophage inhibitory cytokine-1(MIC-1), a divergent member of the TGF β family, does not require a pro-domain for secretion, it does require the pro-domain as a quality control mechanism to ensure proper folding of the mature protein (Bootcov et al., 1997, Proc. Natl. Acad. Sci. USA, 94:11514). As a result, it has been widely believed that all members of the GDNF family require the pro-domain for proper folding or secretion or both.